Hairy Root Transformation Research Articles

A Novel Solid Media-Free In-Planta Soybean (Glycine max. (L) Merr.) Transformation Approach

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation.

Enhancing Agrobacterium-mediated soybean transformation efficiency with an auxiliary solution

Soybean is a crucial source of oil, protein, and biofuel, necessitating efficient transformation systems for advancing research. Agrobacterium-mediated transformation is currently the primary method used in the soybean transformation industry and scientific research. However, the low efficiency and genotype dependency of this technology leave significant room for improvement. This study aimed to enhance soybean transformation efficiency by generating and validating three reporter vectors (ZsGreen, TdTomato, and Ruby) and using Agrobacterium Auxiliary Solution (AAS) containing Silwet L-77 and hormone mixtures. Our findings demonstrate that AAS significantly improves hairy root transformation rates. Specifically, this combination increased total root and cotyledon transformation efficiencies compared to the control. We also found that larger vectors like Ruby reduced transformation efficiency compared to smaller markers like GFP and RFP. Furthermore, AAS slightly reduced the co-transformation rate of two separate vectors compared to single vector transformations. Additionally, AAS enhanced soybean hypocotyl transformation rates across various varieties, consistently increasing positive root and explant efficiencies. Notably, transformation rates varied significantly between varieties, with Forrest differing from Williams 82 and Dongnong 50. This research highlights the importance of auxiliary agents and vector size in optimizing soybean transformation, providing insights for future advancements in genetic modification and biotechnology.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

MiR398b Targets Superoxide Dismutase Genes in Soybean in Defense Against Heterodera glycines via Modulating Reactive Oxygen Species Homeostasis.

MicroRNAs play crucial roles in plant defense responses. However, the underlying mechanism by which miR398b contributes to soybean responses to soybean cyst nematode (Heterodera glycines) remains elusive. In this study, by using Agrobacterium rhizogenes-mediated transformation of soybean hairy roots, we observed that miR398b and target genes GmCCS and GmCSD1b played vital functions in soybean-H. glycines interaction. The study revealed that the abundance of miR398b was downregulated by H. glycines infection, and overexpression of miR398b enhanced the susceptibility of soybean to H. glycines. Conversely, silencing of miR398b improved soybean resistance to H. glycines. Detection assays revealed that miR398b rapidly senses stress-induced reactive oxygen species, leading to the repression of target genes GmCCS and GmCSD1b and regulating the accumulation of plant defense genes against nematode infection. Moreover, exogenous synthetic ds-miR398b enhanced soybean sensitivity to H. glycines by modulating H2O2 and O2- levels. Functional analysis demonstrated that overexpression of GmCCS and GmCSD1b in soybean enhanced resistance to H. glycines. RNA interference-mediated repression of GmCCS and GmCSD1b in soybean increased susceptibility to H. glycines. RNA sequencing revealed that a majority of differentially expressed genes in overexpressed GmCCS were associated with oxidative stress. Overall, the results indicate that miR398b targets superoxide dismutase genes, which negatively regulate soybean resistance to H.glycines via modulating reactive oxygen species levels and defense signals.

Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis)

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

A Highly Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method of Idesia polycarpa and the Generation of Transgenic Plants.

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on host–pathogen interaction.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit’s efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Rapid production of abundant transgenic pomegranate (Punica granatum) hairy roots

Pomegranates (Punica granatum) are known for their high levels of health-beneficial compounds that belong to the hydrolyzable tannin (HT) and flavonoid families. However, a significant gap in our understanding exists of the biosynthetic and regulatory genes related to the accumulation of these compounds. To this end, the induction and transformation of hairy roots presents an exciting opportunity to decipher genetically the functions of candidate genes involved in the HT and flavonoid metabolism. This study aims at improving methods for rapid and abundant hairy root production from pomegranate explants by increasing seed germination rates and expediting the attainment of requisite biomass for multifaceted analyses. We found that treating seeds with sandpaper and concentrated sulfuric acid significantly enhanced their germination rates. Interestingly, no correlation between seed-coat texture and seed germination rate was observed among the 20 pomegranate accessions evaluated in this study. Notably, transferring hairy roots from agar plates to soil promoted rapid root biomass growth compared to maintaining them on agar plates. Root biomass as well as levels of gallic acid (a precursor for HT biosynthesis) and punicalagins (the major HTs in roots) across 15 pomegranate accessions were also analyzed. Three of these accessions exhibited higher seed germination percentages along with augmented biomass and elevated punicalagin levels. These pomegranate accessions emerge as promising germplasm for future genetic transformation and functional genomics studies.

Research on the function of CsMYB36 based on an effective hair root transformation system

ABSTRACT The hairy root induction system was used to efficiently investigate gene expression and function in plant root. Cucumber is a significant vegetable crop worldwide, with shallow roots, few lateral roots, and weak root systems, resulting in low nutrient absorption and utilization efficiency. Identifying essential genes related to root development and nutrient absorption is an effective way to improve the growth and development of cucumbers. However, genetic mechanisms underlying cucumber root development have not been explored. Here, we report a novel, rapid, effective hairy root transformation system. Compared to the in vitro cotyledon transformation method, this method shortened the time needed to obtain transgenic roots by 13 days. Furthermore, we combined this root transformation method with CRISPR/Cas9 technology and validated our system by exploring the expression and function of CsMYB36, a pivotal gene associated with root development and nutrient uptake. The hairy root transformation system established in this study provides a powerful method for rapidly identifying essential genes related to root development in cucumber and other horticultural crop species. This advancement holds promise for expediting research on root biology and molecular breeding strategies, contributing to the broader understanding and improvements crop growth and development.

Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus varieties

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

Injection-based hairy root induction and plant regeneration techniques in Brassicaceae

BackgroundHairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media.ResultsWe initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies.ConclusionWe developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

Study of functional features of plant root systems using CRISPR/Cas-mediated genome editing

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function [1, 2].
 A common construct for efficient genome editing and selection of hairy roots is comprised of three components, i.e., a cassette carrying the gene encoding the Cas nuclease, a cassette expressing the guide RNA (gRNA), and a cassette encoding a screenable or selectable marker [2]. After design and construction, the resulting vector is used to transform plant using appropriateRhizobium rhizogenesstrain.
 Over 26 plant species have been used in experiments combining genome editing and hairy root transformation to date [2]. Possible applications of CRISPR/Cas9 genome editing using hairy root transformation include different directions like test the efficiency of the CRISPR/Cas9 genome editing; obtaining whole genome-edited plants regenerated from individual edited hairy roots; investigation of root development or root function, root nodule symbiosis, resistance to biotic or abiotic stresses, or metabolic engineering [2].
 The basic principles of plant CRISPR/Cas genome editing like the different components of CRISPR/Cas vectors, the types of Cas nuclease, design principles of gRNAs, as well as the possible applications of CRISPR/Cas genome editing in hairy roots will discuss. The application of this method for multigene editing strategy will also be demonstrated onDEEPER ROOTING1genes of cucumber.
 The study was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 075-15-2021-1056).

PsNRT2.3 interacts with PsNAR to promote high-affinity nitrate uptake in pea (Pisum sativum L.)

Nitrate, the primary form of nitrogen absorbed by plants, supplies essential compounds for plant growth and development. Peas are frequently used as rotation crops to improve and stabilize soil fertility. However, the determinants of nitrate uptake and transport in peas remain largely unclear, primarily due to the pea genome's complexity and size. In this study, we utilized the complete genomic information of peas to identify three PsNRT2 family genes within the pea genome. We conducted a comprehensive examination of their protein conserved domains, physicochemical properties, gene structure, and phylogenetic evolution, revealing PsNRT2.3 as the potential key gene for high-affinity nitrate transport in peas. Subcellular localization studies indicated that PsNRT2.3 resides on the plasma membrane. Using hairy root transformation, we noted the predominant expression of PsNRT2.3 in the root stele, which is inducible by nitrate. Our experiments involving overexpression and silencing methods further confirmed that PsNRT2.3 plays a key role in enhancing nitrate uptake in peas. Additionally, our work showed that PsNAR could interact with PsNRT2.3, modulating pea nitrate uptake. After silencing PsNAR, even with the normal expression of PsNRT2.3, the ability of peas to absorb nitrate was significantly reduced. In conclusion, this study identifies the high-affinity nitrate transport gene PsNRT2.3 in peas and clarifies its critical role and regulatory network in nitrate transport, contributing to a new understanding of nitrate utilization in peas.

A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes.

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Fine-tuning CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models.

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9 on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes.

Metabolic variations in root tissues and rhizosphere soils of weak host plants potently lead to distinct host status and chemotaxis regulation of Meloidogyne incognita in intercropping.

Root-knot nematodes (RKNs) inflict extensive damage to global agricultural production. Intercropping has been identified as a viable agricultural tool for combating RKNs, but the mechanisms by which intercropped plants modulate RKN parasitism are still not well understood. Here, we focus on the cucumber-amaranth intercropping system. We used a range of approaches, including the attraction assay, in vitro RNA interference (RNAi), untargeted metabolomics, and hairy root transformation, to unveil the mechanisms by which weak host plants regulate Meloidogyne incognita chemotaxis towards host plants and control infection. Amaranth roots showed a direct repellence to M. incognita through disrupting its chemotaxis. The in vitro RNAi assay demonstrated that the Mi-flp-1 and Mi-flp-18 genes (encoding FMRFamide-like peptides) regulated M. incognita chemotaxis towards cucumber and controlled infection. Moreover, M. incognita infection stimulated cucumber and amaranth to accumulate distinct metabolites in both root tissues and rhizosphere soils. In particular, naringenin and salicin, enriched specifically in amaranth rhizosphere soils, inhibited the expression of Mi-flp-1 and Mi-flp-18. In addition, overexpression of genes involved in the biosynthesis of pantothenic acid and phloretin, both of which were enriched specifically in amaranth root tissues, delayed M. incognita development in cucumber hairy roots. Together, our results reveal that both the distinct host status and disruption of chemotaxis contribute to M. incognita inhibition in intercropping.